Purification and refolding of vascular endothelial growth factor-B.

Vascular endothelial growth factor (VEGF)-A interacts with the receptor tyrosine kinases VEGF-R1 and R2, and the importance of this interaction in endothelial cell (EC) function and blood vessel development has been well documented. Other ligands that interact differentially with these receptors and that are structurally related to VEGF-A include VEGF-B, VEGF-C, VEGF-D, and placenta growth factor (PLGF). Compared with VEGF-A, relatively little is known about the biological role of the VEGF-R1 specific ligand, VEGF-B. Two splice variant isoforms that differ at the COOH-terminus and which retain unique solubility characteristics are widely expressed throughout embryonic and postnatal development. Recent analysis of mice with a targeted deletion of the VEGF-B gene has revealed a defect in heart development and function consistent with an important role in vascularization of the myocardium (Bellomo D et al., 2000, Circ Res 86:E29-E35). To facilitate further characterization of VEGF-B, we have developed a protocol for expression and purification of refolded recombinant protein from Escherichia coli inclusion bodies (IBs). The approach developed resolves a number of significant issues associated with VEGF-B, including the ability to heterodimerize with endogenous VEGF-A when co-expressed in mammalian cells, a complex secondary structure incorporating inter- and intrachain disulfide bonds and hydrophobic characteristics that preclude the use of standard chromatographic resins. The resulting purified disulfide-linked homodimer was demonstrated to bind to VEGF-R1 and to compete with VEGF-A for binding to this receptor.

Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells.

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50-500 nM, and was abolished by the PKC inhibitor, Ro 31-8220 (10 microM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 microM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca2+. The phorbol ester cyclic AMP response was additive with that to K+ depolarisation, and synergised with those to prostaglandin E1 and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or Gs.

[Sensitivity and specificity of transrectal ultrasonography in the preoperative staging and postoperative follow-up in rectal neoplasms. Experience with 100 clinical cases]. / Sensibilità e specificità dell'ultrasonografia trans-rettale nello staging preoperatorio e nel follow-up post-chirurgico delle neoplasie del retto. Esperienza su 100 casi clinici.

UNLABELLED: Preoperative staging plays an important role in the surgical treatment of rectal cancer. The most sensitive imaging techniques currently available are CT, MRI and transanal ultrasound (TAUS). The aim of the study was to evaluate the sensitivity and specificity of TAUS in the preoperative staging and postoperative follow-up of rectal cancer. METHODS: From January 1992 to May 1999, TAUS was used to study 100 patients with rectal cancer. RESULTS: Patients were staged according to the Astler-Coller classification: 1) A: 8 patients; 2) B1: 16 patients; 3) B2: 22 patients; C1: 30 patients; C2: 24 patients. The sensitivity and specificity of TAUS in the preoperative staging of these tumors were 96% for the T parameter, and 100% for the N parameter. The N but not the T parameter results are in line with the values reported in the international literature. CONCLUSION: Transanal ultrasound, in our personal experience, has proved to be a very accurate imaging technique in the preoperative staging and postoperative follow-up of rectal cancer.

[Minimally invasive percutaneous ultrasonography-guided treatment of postoperative splenic abscess. Personal experience with 5 clinical cases]. / Terapia mininvasiva percutanea ultrasonoguidata degli ascessi post-chirurgici della milza. Esperienza personale su cinque casi clinici.

INTRODUCTION: Splenic abscesses are very rare pathologies encountered in daily clinical practice. The treatment, after failure of other medical treatments, has basically been surgical, in spite of large diffusion of percutaneous echo and CT techniques. The initial hesitation in applying this sort of method was caused by an unfounded fear of difficult management of haemorrhagic complications. METHODS: In our Clinic we treated 6 postoperative abscesses, observed in 5 patients. One of these (16%) was double and 3 concomitant with other intraperitoneal abscessual septic collections, which received priority in percutaneous treatment. In four cases (67%), Van-Sonnemberg drainage catheters were placed which caliber varied from 12 to 14 F. In the remaining two cases, we evacuated the abscess using an echo-guided needle puncture. In two cases we applied fibrinic glue; in one case as a haemostatic agent and in the other, to protect the wide abscessual cavity. RESULTS: We recorded the resolution of the symptomatology and instrumental findings in all patients. One patient died two weeks later due to unrelated causes. We recorded one minor complication which involved an intra-abscessual haemorrhage, which was then treated by fibrinic glue (Tissucol). CONCLUSION: Echo-guided percutaneous treatment, at present, is the treatment of choice for both multiple or single splenic abscesses. This treatment is not used for the miliaric form because we believe that in this case, the most appropriate choice is intensive medical treatment, which if fails, may require splenectomy.

[Palliative treatment of neoplasms of the bilio-pancreatic area. Surgical diversion treatment versus endoscopic-percutaneous approach]. / Trattamento palliativo delle neoplasie del distretto bilio-pancreatico. Terapia derivativa chirurgica versus approccio endoscopico-percutaneo.

Bilio-pancreatic (BPD) tumours are pathologies with an overall resectability rate of 20-25%, with a high percentage of patients directed towards palliative treatment (70-90%). One hundred and twenty-eight patients underwent palliative treatment for BPD tumours. The M:F ratio was 1:1.5 and the mean age 61 years. The tumours were topographically divided into: 84 (63%) localised in the pancreatic head region; 34 (25%) biliary duct tumours and 6 arising in the ampulla of Vater. 42 patients (30%) underwent palliative surgical treatment (group 1) and 86 (70%) received non-surgical treatment (endoscopic percutaneous) (group 2). We also performed 6 gastrointestinal by-passes (GEA). The early morbidity rate was 36% in the first group and 12.8% in the second. The incidence of late morbidity in the first group was nil, with a mean survival of 10 months. Mortality was nil in both groups. Surgical by-pass is advisable in the presence of patients with a longer life expectancy and in good clinical condition but presents a high rate of early morbidity and a low percentage of late complications. The authors conclude that validity of the methods is similar. They believe that, in selected cases, surgical treatment remains the therapy of choice for BPD tumours.

[Carcinomas of the digestive system in patients under 20 years old]. / I carcinomi dell'apparato digerente in pazienti di età inferiore a 20 anni.

Gastrointestinal cancers are very scarce for the people under twenties. Such a rarity, as misguides diagnostic trend, is responsible, in the greater part of cases, of the lag in the recognizing these neoplasm and, consequently, reduces the expectations of surviving whether medium or long term. Authors report their experience as regards to observation of two cases of malignant neoplasm of the stomach and of the recto-sigmoid joint in young patients of fourteen and nineteen years old and they emphasize the importance of a complete diagnostic route which, in spite of age, does not exclude the possibility of a neoplastic pathology in the presence of suggestive persistent symptomatology. It is underlined the importance of early diagnosis in fact survival yield is always and however dependent on a disease stage and not on age. Furthermore it is underlined that the young patient is subject to a rate of postoperative complications which is marginal compared with intervention yield, this is translated in a better responsiveness of young patient to radical and, if it is necessary, extended interventions.

[Surgical treatment of acute colonic diverticulitis. Personal experience]. / Il trattamento chirurgico della diverticolite acuta del colon. Esperienza personale.

BACKGROUND: Colonic acute diverticulitis presents in patients that suffered from colonic diverticula in 10-25% of cases higher percentage in the elder patient. Colonic acute diverticulitis mortality leads high rates in over 70 years old patients with a value a upper than 75-80%. Perforation and diffuse peritonitis are the more serious complications. Accurate clinical staging followed by correct surgical option are the base of the therapeutical success. METHODS: Between january 1995 and june 2000 we studied by a retrospective analysis 56 patients (34M/22F), mean age 67, hospitalized cause acute perforate colonic diverticulitis, all patients were recorded in order their clinical conditions APS score and Hinchey staging of the disease. At the time of the recovery all patients underwent at clinical examination, abdominal ultrasound, plane X-ray, and bloody tests. Surgical operations performed were: PRA; PRA and protective colostomy; Hartmann procedure. In the patients with APS score > 6-10 and aged > 70 Hartmann procedure was performed. RESULTS: Overall mortality was 9% (5 pazienti), overall morbility was 40%. On the basis of the I and II Hinchey stage mortality rate was 0%. In the III stage was recorded mortality of 4% and 33% in IV stage. Complication were: 6 anastomotic lekage; 7 infections of the laparotomic wound; 1 incisional hernia; 2 lekeages of the stomoa; 3 stomatitis; 5 proctitis. CONCLUSION: Therapeutical choise treatment has to consider Hinchey stage and APS score. Hartmann procedure is indicated in stage III-IV with score APS > 8-10. For the other patients primary resection anastomosis and if is necessary associated colostomy is the treatment recommended.

[Periampullar tumors: role of intraoperative color-Doppler ultrasonography in the evaluation of vascular invasion. Methods available to the surgeon and personal experience]. / I tumori della regione periampollare: ruolo della ultrasonografia color-doppler intraoperatoria nella valutazione della invasione vascolare. Metodiche a disposizione del chirurgo ed esperienza personale.

BACKGROUND: Tumors of the head of the pancreas and of the periampullary region, similar nosologic entity, represent the fourth most common cause of cancer death. These tumors are characterized bt high mortality rate associated with a low percentage of surgical resectability. Vascularly involvement is, in our experience, one of the criteria of exclusion for curative surgical resection. For this reason regional pancreasectomy don't show, in several case records, comforting datas about survival. METHODS: From 1995 to 1998 we introduced in the operative practice the color--doppler ultrasonography to diagnose vasculary involvement. We evaluated twenty patients that previously were candidated for surgical therapy consisting in a duodenal-pancreasectomy. All the patients were staged by preoperative abdominal ultrasound, E.R.C.P., CT, and angio M.R.I.. In no one of these patients were founded signs of vascularly involvement. We recognized by intraoperative color-doppler ultrasound three microscopic invasions of spleno-mesenteric-portal vasculary axis. RESULTS: U.S. I.O. C.D. in our case reports has underlined a sensibility and specificity of 100%. It has discovered, also, a false positive for neoplastic thrombosis, and a case of false negative. CONCLUSIONS: Intraoperative ultrasound shows a high sensibility about the intraoperative staging for the bibliopancreatic tumors. The study about the possible vascularly involvement is helped by the application of the colour in terms of an high sensibility and diagnostic care.

[Short- and long-term monitoring with high-resolution ultrasonography of postoperative thyroid residue. Personal experience with 200 thyroidectomies]. / Monitoraggio a breve e lungo termine mediante ultrasonografia ad elevata risoluzione (us-hr) del residuo tiroideo post-operatorio Esperienza personale su 200 tiroidectomie.

BACKGROUND: After having abandoned conservative operations of the thyroid (partial resection, enucleal resection) today total thyroidectomy and sub-total thyroidectomy and near total thyroidectomy are the operations of choice. Cause the frequent post-operative scintigraphic signs, on the 3rd month, of thyroidal residue of significative size, founded in our patients operated for sub-total thyroidectomy, we begun to study the echografical monitoring of the thyroidal recess. METHODS: From January 1995 to December 1997 we operated 200 patients for Thyroidectomy. The kinds of operations were: total thyroidectomy for 96 patients (48%), sub-total thyroidectomy for 64 patients (32%), and near total thyroidectomy for 40 patients (20%). We controlled all the patients by ecografical monitoring of the thyroidal recess to detect and to determinate the possible thyroidal residue, that it will be monitorized on the 7th day, on the 1st month, on the 6th month and on the 12th month. We measured the thyroidal residual dimensions and volume. We recorded all the examinations by VCR or film and after we elaborated the results by exponential graphics. The patients that showed the thyroidal residues underwent to a scintigraphy. RESULTS: The thyroidal residuals were measured within the 7Th post-operative day. In the following controls we have noted a thyroidal residue hypertrophy. Multinodular goither was the disease with the highest degree of growth (until the 200% of the first measure of the residue). After an initial phase of growth almost all the patients showed a trend to the stabilization, with the exception only of the patient that suffered from multinodular goither. The scintigraphic examination was not realiable, in our experience, about the mathematic measurement of the thyroidal tissue, showing always an overvaluation of the residue. The best specificity was showed by US-HR. CONCLUSIONS: The patient that undergoes to total thyroidectomy needs an adequate suppressive and substitutive therapy. We identify the presence of several stimulating factors the growth of the residual thyroidal tissue that could arise to promote the hyperplasia and the hypertrophy of the parenchyma (EGF, TSG, TSI, PG). There are factors that are unidentified and are not subject to the suppression caused by substitutive therapy.

Shared synteny between human chromosome 10 and chromosome 1 of the marsupial tammar wallaby, Macropus eugenii.

Marsupial homologs of the human chromosome 10 loci IL2RA, HK1, and PLAU have been cloned and mapped by fluorescence in situ hybridization to chromosome 1q of the tammar wallaby, Macropus eugenii. Relative distance measurements of the hybridization signals on M. eugenii chromosome 1 show that marsupial homologs of human (HSA) 10p IL2RA and 10q HK1/PLAU flank the marsupial homologs of the human 5q gene IL5 and the human 15q imprinted genes SNRPN and ZNF127. The shared synteny, therefore, does not necessarily mean that HSA 10 represents an ancestral grouping; rather, it suggests that HSA 10p and HSA 10q represent two different ancestral mammalian units which fused directly in primates and were incorporated independently into two different regions of the same chromosome in marsupials.

Isolation and characterization of marsupial IL5 genes.

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.

The evolution of human chromosome 21: evidence from in situ hybridization in marsupials and a monotreme.

We have mapped five human chromosome 21 (HSA 21) markers in marsupials and a monotreme, two major groups of mammals that diverged from eutherians 130-150 and 150-170 million years before present (MYrBP), respectively. We have found that these genes map to two distinct autosomal sites, one containing SOD1/CBR/BCEI and the other containing ETS2/INFAR, in the marsupials Macropus eugenii and Sminthopsis macroura (which belong to orders that diverged 40-80 MYrBP), as well as in the monotreme Ornithorhynchus anatinus (the platypus). Since marsupials and monotremes diverged independently from eutherians, these data suggest that HSA 21 genes were originally located in two separate autosomal blocks. In another Sminthopsis species, SOD1 is linked to TRF (a marker on HSA 3q), suggesting that the ancestral SOD1/CBR/BCEI region also included HSA 3 markers. We suggest that these blocks became fused early in the eutherian evolution to form a HSA 3/21 chromosome, which has remained intact in artiodactyls, but has been independently disrupted in both the primate and rodent lineages.

Shared DNA sequences between the X and Y chromosomes in the tammar wallaby - evidence for independent additions to eutherian and marsupial sex chromosomes.

Marsupial sex chromosomes are smaller than their eutherian counterparts and are thought to reflect an ancestral mammalian X and Y. The gene content of this original X is represented largely by the long arm of the human X chromosome. Genes on the short arm of the human X are autosomal in marsupials and monotremes, and represent a recent addition to the eutherian X and Y. The marsupial X and Y apparently lack a pseudoautosomal region and show only end-to-end pairing at meiosis. However, the sex chromosomes of macropodid marsupials (kangaroos and wallabies) are larger than the sex chromosomes of other groups, and a nucleolus organizer is present on the X and occasionally the Y. Chromosome painting using DNA from sorted and microdissected wallaby X and Y chromosomes reveals homologous sequences on the tammar X and Y chromosomes, concentrated on the long arm of the Y chromosome and short arm of the X. Ribosomal DNA sequences were detected by fluorescence in situ hybridization on the wallaby Xp but not the Y. Since no chiasmata have been observed in marsupial sex chromosomes, it is unlikely that these shared sequences act as a pseudoautosomal region within which crossing over may occur, but they may be required for end-to-end associations. The shared region of wallaby X and Y chromosomes bears no homology with the recently added region of the eutherian sex chromosomes, so we conclude that independent additions occurred to both sex chromosomes in a eutherian and macropodid ancestor, as predicted by the addition-attrition hypothesis of sex chromosome evolution.

Localization of four human chromosome 21 genes--SOD1, ETS2, IFNAR, and CBR--to two different chromosomes in the marsupial species Macropus eugenii.

We have mapped the chromosomal location of four genes previously assigned to human chromosome 21--Cu/Zn superoxide dismutase (SOD1), the protooncogene ETS2, the interferon alpha/beta receptor gene (IFNAR), and the carbonyl reductase gene (CBR)--in the tammar, Macropus eugenii. The genes are localized on two separate autosomes: SOD1 and CBR map to chromosome 7 and ETS2 and IFNAR map to chromosome 3 or 4. These results provide the first example of asynteny between SOD1/CBR and ETS2/IFNAR in a mammalian species. The results suggest that either this synteny group has been disrupted in the marsupial lineage, or, alternatively, the genes located on human chromosome 21 may have been joined after the marsupials diverged from the eutherian mammals some 130-150 million years ago.

Human vascular endothelial growth factor B: characterization of recombinant isoforms and generation of neutralizing monoclonal antibodies.

1. The vascular endothelial growth factor (VEGF) family is a focus of interest with respect to novel therapies for cardiovascular disease. Members of this family bind differentially to three receptor tyrosine kinases, namely VEGF-R1, -R2 and -R3, and to the semaphorin receptors neuropilin 1 and 2. The role of VEGF-R1 and the factors that interact exclusively with this receptor (VEGF-B and placenta growth factor) has remained controversial. 2. To further elucidate the role of VEGF-B in blood vessel formation and function, we have expressed, purified and refolded both naturally occurring VEGF-B isoforms and a truncated amino acid 10-108 form. All refolded proteins have been demonstrated to bind to VEGF-R1 with appropriate kinetics in biosensor-based analysis. 3. Robust cell assays for VEGF-R1 ligands, such as VEGF-B, have been problematic. We have developed an assay based on a chimeric receptor consisting of extracellular domains 1-4 of VEGF-R1 and the transmembrane and intracellular domains of gp130. The cell line expresses luciferase to high levels 24 h after exposure to VEGF-A and both refolded VEGF-B167 and the short 10-108 isoform have been demonstrated to be active in this assay. 4. The novel cell-based assay, in combination with a variety of immunochemical approaches, has been used to identify and characterize monoclonal antibodies that neutralize VEGF-B activity.